Design And Synthesis Of Multi-recognition Fluorescent Probes And Their Biological Applications

A single recognition fluorescent probe can only recognize one kind of analyte,and cannot be used to study the association and synergistic mechanism between multiple active substances.There are obvious defects in the use of two or more single-recognized fluorescent probes for multifunctional detection.For example,when two or more single-recognized fluorescent probes are used for monitoring at the same time,there is potential crosstalk;because of their different subcellular localization or photobleaching rate,the imaging situation will be complex.The multifunctional fluorescent probes can recognize multiple active substances,which are accompanied by the change of fluorescence and the change of solution color.Generally,multichannel fluorescence response can be observed in cell imaging.Due to the above-mentioned advantages,multifunctional fluorescent probes have attracted widespread attention in recent years.They have been used to simultaneously identify active substances with similar structures,visually monitor the interaction between different active substances,and study the mechanism of action of different active substances.Based on this,in this paper,three redox-responsive multifunctional fluorescent probes?probe NPCC,probe NPCF,and probe NPCl A?and two non-redox-responsive multifunctional fluorescent probes?probe NPHSHNO and probe NPCys HS?have been developed,the details are as follows:?1?The first endoplasmic reticulum-targeted two-photon fluorescence probe NPCC was designed and synthesized for sequential detection of Cys,GSH and ClO^–.The study of spectral properties showed that the probe had good selectivity,high sensitivity,low toxicity,and could dynamically distinguish Cys from GSH.Cell imaging studies displayed that NPCC could be used for two-photon imaging of intracellular ClO^–.The probe could be localized in the endoplasmic reticulum,and could be used to monitor reductive stress in the endoplasmic reticulum.In addition,the probe could distinguish cancer cells from normal cells.The probe can be used as a simple and effective tool to study the relationship between RSS and ROS.?2?A near-infrared fluorescent probe?NPCF?was exploited for dual-recognition of sulfur dioxide derivatives and pH with“naked-eye”in 100%buffer solutions.NPCF showed good selectivity and high sensitivity?LOD 22.7 nM?for HSO₃^-.With pH changing from 9.21 to 4.26,the fluorescence of NPCF showed a significant increase at 610 nm?up to 885-fold?.NPCF was successfully used in zebrafish experiments for detecting HSO₃^-,which indicated that NPCF had potential application value in biology.Using NPCF,we found that SO₂ could reduce inflammation caused by LPS and reduce acidification environment.At different stages of cell injury and apoptosis induced by CCCP,the changes of SO₂ and pH value were different.In the initial stage of injury and apoptosis,the amount of SO₂ would gradually increase,and the pH would gradually decrease;in the stage of severe injury,the amount of SO₂ would gradually decrease,and the pH would gradually increase.This study provided a new idea,a simple and effective tool for understanding the mechanism of cell injury.In addition,NPCF could be used to monitor the level of SO₂ and the change of pH value in cells to distinguish normal cells from cancer cells.The probe is expected to be a simple and practical tool for early cancer diagnosis.?3?A two-photon fluorescent probe NPCl A with two-site recognition,three-channel imaging and mitochondrial targeting,was designed and synthesized.NPCl A could dual-recognize HSO₃^–and ClO^–with three-band fluorescence changes,and had the advantages of“naked eye”recognition,good selectivity,and high sensitivity.Using this probe to monitor the levels of HSO₃^–and ClO^–in cells could distinguish normal cells from cancer cells,which indicated that NPCl A had potential application value in the early diagnosis of cancer.Excessive HSO₃^–and LPS-induced mitochondrial stress experiments showed that SO₂ and ClO^–were in a dynamic equilibrium.In addition,we also performed in situ cell imaging experiment of excess ClO^–-induced oxidative stress,and the results showed that cellular stress produced endogenous SO₂.This probe further explained the dual oxidation/antioxidation effect of SO₂ in mitochondria.The probe provided a useful detection tool for monitoring the balance of ROS/RSS during mitochondrial stress and understanding the dual effects of oxidation/antioxidation of SO₂ in mitochondria.?4?The first dual-recognition fluorescent probe NPHSHNO that recognized both HS^–and HNO was designed and synthesized.The probe consisted of one fluorophore and two recognition sites.During the recognition process of NPHSHNO,PET and ICT occurred.NPHSHNO had significant color and fluorescence changes,and could selectively detect HS^–and HNO,with detection limits of 1.02?M and 0.67?M,respectively.The two recognition sites of NPHSHNO recognized HS^–and HNO,respectively,which could be used as two input signals of the molecular logic gate.Only HS^–and HNO were presented at the same time,the“AND”logic gate signal was released and the probe's fluorescence was completely released.NPHSHNO was successfully applied to the detection of HS^–and HNO in cells.?5?A multifunctional fluorescent probe NPCys HS that recognized both Cys and HS^–was designed and synthesized.The probe consisted of one fluorophore and two recognition sites.“NBD-N-”as the recognition site of HS^–,could distinguish Cys and HS^–in red channel,and the introduction of“NBD-O-”allowed NPCys HS to distinguish Cys and HS^–in green channel.During the recognition process,the PET mechanism and the ICT mechanism occurred.In the process of identification,there were obvious changes in solution color and fluorescence intensity.NPCys HS had good selectivity,with detection limits of 0.15?M and 37.7 n M,respectively.NPCys HS was successfully applied to the detection of Cys and HS^–in cells.